(Investigative Ophthalmology and Visual Science. 2000;41:290-293.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
A New Leu253Arg Mutation in the RP2 Gene in a Japanese Family with X-Linked Retinitis Pigmentosa
Yuko Wada1,
Mitsuru Nakazawa2,
Toshiaki Abe1 and
Makoto Tamai1
1 From the Department of Ophthalmology, Tohoku University School of Medicine, Sendai, Japan; and the
2 Department of Ophthalmology, Hirosaki University School of Medicine, Hirosaki, Japan.
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Abstract
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PURPOSE. To identify the clinical findings in a Japanese family with X-linked
retinitis pigmentosa associated with mutation in codon 253 (Leu253Arg)
in the RP2 gene.
METHODS. Case reports included clinical features and results of fluorescein
angiography, electroretinogram, kinetic visual field testing, and DNA
analysis. Two affected hemizygotes with retinitis pigmentosa associated
with transversion mutations in codon 253 (Leu253Arg) of the RP2 gene
and the obligate carriers were examined.
RESULTS. A novel Leu253Arg mutation of the RP2 gene was found to cosegregate
with retinal degeneration in two affected males and two carriers in
female heterozygote in a Japanese family. The ophthalmic findings in
hemizygote showed severe retinal degeneration. In the obligate carrier,
mild chorioretinal degeneration was observed in both eyes but a
tapetal-like reflex of the fundus was not apparent.
CONCLUSIONS. The mutation at codon 253 of the RP2 gene is the first mutation
reported in a Japanese family. It is concluded that the mutation of the
RP2 gene also causes the X-linked retinitis pigmentosa in Japanese
patients.
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Introduction
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Retinitis pigmentosa (RP) is a progressive retinal degeneration
with bone spicules and shows genetic heterogeneity, with autosomal
dominant, recessive, and X-linked forms. The X-linked form of RP is the
most severe, and patients show partial or total blindness by the third
or fourth decade.1
In our population of patients with RP, X-linked RP (XlRP) comprises
approximately 2% of patients with RP. The ratio of X-linked to
non–X-linked RP in Japan is lower than that in other European
countries.
In 1984, the X-linked form of RP (RP2) was mapped to
Xp11.3,2
and another X-linked RP gene (RP3) was mapped to
Xp21.3
Positional cloning of the RP3 gene was performed in 1996 and found to
encode a putative guanine nucleotide exchange factor.4
It
was discovered that mutations in this gene are in less than 20% of the
patients with XlRP. In 1998, RP2 gene, which showed homology with human
cofactor C, which was protein involved in the ultimate step of
ß-tubulin folding, was positionally cloned, and six mutations in the
RP2 gene were discovered in European patients with X-linked
RP.5
Furthermore, five mutations in the RP2 gene were
reported for the XlRP families in a North American
cohort.6
We describe herein the ocular features associated
with a newly identified RP2 gene mutation in a Japanese family with
XlRP. The aim of this study was to assess the phenotypic manifestation
associated with this mutation and the frequency of RP2 mutation in
Japanese patients with XlRP.
The mutation gave rise to a thymine to guanine transversion in the
second nucleotide at codon 253, resulting in a substitution of an
arginine residue for a leucine at the codon. The phenotypic expression
produced by this mutation is characterized by severely accelerated
retinal degeneration. The present report is the first to describe the
clinical features associated with an RP2 mutation.
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Methods
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Subject and Mutation Analysis
We screened 39 genomic DNA samples isolated from 25 patients with
XlRP and 14 carriers to search for mutations in the RP2 gene by means
of nonradioisotopic single-strand conformation polymorphism (SSCP) with
a modification previously described.7
We further screened
150 control X-chromosomes with this gene. Genomic DNA was isolated from
leukocytes prepared from a sample of the patient’s blood (10–15 ml),
using a protocol previously described.8
The sequence from exon1 to exon5 of the RP2 gene was amplified by
polymerase chain reaction. Eight sets of oligonucleotide primer pairs
were prepared to cover the application of these sequences. The
polymerase chain reaction products were analyzed by SSCP analysis.
After electrophoresis, DNA bands were visualized by silver staining.
The mutation or polymorphism was detected by the presence of abnormal
bands derived from a mutant allele.
One pedigree was the focus for this study (Fig. 1)
, which was identified as having a Leu253Arg mutation. None of 150
control X-chromosomes had this mutation. Furthermore, we screened the
RPGR gene to search for other mutations on our families, and no
mutation was found in the RPGR gene. The tenets of the Declaration of
Helsinki were followed, and informed consent was obtained from all
subjects who participated in this study.
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Figure 1. A pedigree showing affected members with XlRP associated with Leu253Arg
mutation in the RP2 gene. Open symbols indicate
unaffected subjects; solid symbols, affected;
X, individuals examined in this study; dot in the
circle, carrier; symbols with slashes, deceased
member; and arrow, proband.
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Clinical Examination
We examined the two affected patients and the carriers of
this pedigree at Tohoku University Hospital (Sendai, Japan). The
ophthalmic examinations included best corrected visual acuity,
slit-lamp biomicroscopy, kinetic visual field examination, fundus
examination, and electroretinograms (ERGs). Ophthalmoscopic findings
were recorded by color fundus photography. Fluorescein angiography was
performed for one affected patient. Kinetic visual field examination
was performed with a Goldmann perimeter with V-4-e and III-4-e
isopters. The ERG testings were obtained using a single flash or 30-Hz
flicker stimulus of red light under light-adapted conditions for
cone-isolated responses, a dim blue flash in the dark-adapted condition
(30 minutes) for rod-isolated responses, and a bright white flash (20
J) in the dark-adapted state for recording maximal responses of both
rods and cones. These tests were performed under controlled conditions
that conform to the standard of the International Standardization
Committee.9
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Results
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Ophthalmologic Examination
The proband (III-4), a 29-year-old man, realized early in the
first decade of life that he had impaired night vision and visual
acuity. When he was 6 years old, he was diagnosed by a nearby
ophthalmologist as having RP. The patient (III-1) was diagnosed as
having RP in his early teens by the local ophthalmologist. He could not
be referred to our hospital because he lived in a distant place and was
restricted because of the progressive retinal degeneration. The
proband’s visual acuity was corrected to 0.2 OD with a -6.5 diopter
sphere and 0.06 OS with -9.00 to 1.00 x 180 refraction. Fundus
examination showed bilateral pigmentary retinal degeneration and
attenuation of retinal arteries. Fluorescein angiography disclosed
hyperfluorescence from the posterior pole to the peripheral retina that
corresponded to the mottled retina, suggesting atrophic changes in the
retinal pigment epithelial layer (Fig. 2)
. Goldmann kinetic visual field testing showed severely constricted
central visual field for V-4-e target and constricted oval visual field
in the lower area (Fig. 3)
. The ERG testing, a bright white flash in the dark-adapted state,
showed a nonrecordable response.
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Figure 2. Fundus photograph from the proband with Leu253Arg mutation and the
obligate carrier. The right eye of the proband (top
left) and the right eye of the carrier (top
right). Fluorescein angiography of the proband. Both eyes had
hyperfluorescent spots corresponding to the atrophy of retinal pigment
epithelium. The right eye (bottom left) and the left eye
(bottom right).
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The two carriers had no visual complaints. The carrier’s (II-4)
corrected visual acuity was 0.8 OD with -2.5 diopter sphere and 0.7 OS
with -3.0 diopter sphere. Slit-lamp biomicroscopy disclosed a
normal-appearing cornea, anterior chamber, iris, lens, and vitreous in
each eye. A mild change of retinal pigment epithelium in the posterior
pole was detected in the II-4 carrier, but no retinal change was seen
in the other carrier (II-3; Fig. 2 ).
DNA Analysis
The results of nonradioisotopic SSCP analysis of exon2
showed that a mutant band was observed in the proband, and both the
mutant band and the normal band were observed in his mother, which
showed that she was the obligate carrier (Fig. 4)
. The subsequent nucleotide sequence disclosed that the proband had a
transversion change of thymine to guanine in the second nucleotide at
253 and his mother had both normal and abnormal sequences. This
alteration causes an amino acid substitution of arginine residue for
leucine residue in codon 253 of the RP2 protein (Fig. 4)
. To confirm
that this novel Leu253Arg mutation is a disease-causing mutation, we
further screened the other affected patient, the carrier, and non
affected family members with the RP2 gene by direct sequencing. The
Leu253Arg mutation cosegregated with the disease in the pedigree and
was not detected in other 24 XlRP patients and150 control
X-chromosomes.
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Figure 4. Results of SSCP analysis (top) and DNA sequence analysis
(bottom) of a family with the Leu253Arg mutation in the
RP2 gene. Normal control is designated as N. Lane 8
indicates the obligate carrier (II-4); lane 9, the
proband (III-4). Lanes 1 to 7 and
lane 10 to lane 11 indicate the other
XlRP patients. Identical abnormal band shifts (arrows)
are seen in both cases; furthermore, the carrier had normal band.
Arrowheads indicate the position of the normal allele
and arrows show the position of the mutated alleles.
Bottom: the sequence from the carrier (II-4) showed both
normal alleles and mutant allele in codon 253. Arrows
indicate the position of the mutation. In the lower sequence, mutant
allele from the affected patients shows CGA in codon 253, resulting in
the substitution of arginine for leucine.
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Discussion
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No previous reports regarding phenotypes associated with
RPGR gene or RP2 gene have been shown in Japanese patients with XlRP.
In fact, only 12 mutations in the RP2 gene have been reported, and 1 of
these mutations was a missense mutation and the rest were nonsense
mutations.5
6
Furthermore, the clinical features of XlRP
associated with the mutation in the RP2 gene have not been reported. In
this report, we first described the ocular findings with the Leu253Arg
mutation in the RP2 gene in a Japanese patient with XlRP. This mutation
within exon2 occurred outside the region homologous to a cofactor C,
where most of the reported mutations have been found. Although the
precise effect of the Leu253Arg change on the protein product is still
unclear, we can assume that a positive charge of arginine residue
instead of a hydrophobic leucine residue can impede, to some extent,
physiological structure and function of the RP2 protein.
The ocular finding in the affected male showed a severe form of
RP; he had an impairment of night vision and a
deterioration of central vision within the first two decades of life.
These findings are similar to those in patients who had mutations in
the RPGR gene10
11
and in the RP2 gene.5
Recently it was reported that mutations in the RPGR gene and the RP2
gene were found in approximately 20% and 18%, respectively, of the
patients with XlRP.5
12
In our study, we found mutations
in approximately 4% of Japanese patients with XlRP, which implies that
RP2 mutations are less frequent in Japanese patients than in European
and American patients. One explanation of the low frequency of the
mutation in the RP2 gene may be an ethnic difference.
This novel Leu253Arg mutation, which we first detected in a Japanese
patient with XlRP, supports the theory that mutation in the RP2 gene
causes XlRP in Japanese patients. In the present study, we did not
detect nonsense mutation in RP2 gene, we could not examine the
difference between the clinical features caused by the missense
mutation and those by the nonsense mutation. Therefore, additional
families with XlRP must be studied for mutations to ascertain the
phenotype–genotype correlation in the RP2 gene.
From a clinical point of view, further correlations between specific
mutations and their phenotypes are needed to augment our understanding
not only of the molecular mechanism of diseases but also of diagnostic
and prognostic values.
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Footnotes
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Supported in part by a grant from the Research Committee on Chorioretinal Degenerations and Optic Atrophy, the Ministry of Health
and Welfare of the Japanese Government (Dr. Tamai; Tokyo, Japan); and a Grant-in-Aid for Scientific Research from the Ministry of Education,
Science, and Culture of the Japanese Government (Dr. Tamai, A-2-10307041), Tokyo, Japan.
Submitted for publication March 2, 1999; revised June 18, 1999; accepted August 2, 1999.
Commercial relationships policy: N.
Corresponding author: Yuko Wada, Department of Ophthalmology, Tohoku University School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai 980-77, Japan. yukow{at}oph.med.tohoku.ac.jp
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Top
Abstract
Introduction
